J Mol Biol, 1983 Dec 15, 171(3), 263 - 79 Escherichia coli phenylalanyl-tRNA synthetase operon is controlled by attenuation in vivo; Springer M et al.; The two subunits of phenylalanyl-tRNA synthetase are made from two adjacent, cotranscribed genes that constitute the pheS,T operon . Three different fusions between pheS,T and lac genes were constructed in order to study the regulation of the pheS,T operon in vivo . We show, using these fusions, that phenylalanyl-tRNA synthetase transcription is derepressed when the level of aminoacylated tRNAPhe is lowered by mutational alteration of the synthetase . The pheS,T operon is also derepressed in strains carrying a trpX mutation . The gene trpX codes for an enzyme that modifies both tRNATrp and tRNAPhe and a mutation in that gene causes derepression of the trp and pheA operons, both of which are controlled by attenuation . The in vivo features of the regulation of pheS,T expression described here in correlation with the DNA sequence and in vitro transcription results described in the accompanying paper by Fayat et al . indicate that phenylalanyl-tRNA synthetase is controlled by attenuation in a way analogous to several amino acid biosynthetic operons. J Mol Biol, 1983 Dec 15, 171(3), 239 - 61 Escherichia coli phenylalanyl-tRNA synthetase operon region . Evidence for an attenuation mechanism . Identification of the gene for the ribosomal protein L20; Fayat G et al.; The nucleotide sequences of pheS and of the beginning of pheT have been determined . The genes pheS and pheT code, respectively, for the small and large subunits of phenylalanyl-tRNA synthetase, an alpha 2 beta 2 enzyme . Upstream from pheS the sequence shows another open reading frame of 354 nucleotides (rplT), which accounts for a protein of Mr 13,400 . The product of this gene, previously named "P12", is identified as the ribosomal protein L20 . The promoter for the pheS, T operon was located 368 nucleotides in front of pheS by transcription experiments in vitro . The promoter site is followed by a short open reading frame, which codes for a 14-residue peptide containing five phenylalanine residues . Immediately downstream from the stop codon of this open reading frame, the DNA sequence indicates that the transcript can be folded into three alternative secondary structures, one of which is a site of transcription termination . In vitro, 90% of transcription products initiated at the pheS, T promoter terminate at this site . However, long run-off transcripts proceeding through the terminator and covering the pheS structural gene are observed . No other transcription initiation could be detected between the terminator and the pheS structural gene . All these results are consistent with a mechanism by which phenylalanine-mediated attenuation controls the expression of phenylalanyl-tRNA synthetase . Further evidence is provided for this model by the features of pheS, T regulation in vivo (see the accompanying paper). Eur J Biochem, 1983 Dec 15, 137(3), 573 - 80 Identification of the gene for DNA helicase II of Escherichia coli; Taucher-Scholz G et al.; Using a modification of the solid-phase radioimmune assay of Broome and Gilbert {Proc . Natl Acad . Sci . USA, 75, 2746 (1978)} to screen the plaques of lambda recombinant phages for the presence of an elevated level of helicase-II-specific antigen, we have identified the gene for helicase II in a library of Escherichia coli DNA . The DNA selected was subcloned from lambda into plasmid vectors; restriction analysis located the DNA region encoding helicase II in a PvuII fragment identical in size (2900 base pairs) and restriction pattern to that which contains the uvrD gene . Plasmids carrying this DNA fragment complemented the increased sensitivity to ultraviolet irradiation and the mutator phenotype of uvrD mutants . Furthermore, uvrD502 mutant cells were found to liberate no helicase II activity upon extraction . Following transformation with the cloned DNA, active helicase II was recovered from the mutant cells . These results support the view that helicase II is encoded by uvrD. Biochim Biophys Acta, 1983 Dec 12, 749(2), 198 - 203 Distinct metal cofactor-induced conformational states in the NAD-specific malic enzyme of Escherichia coli as revealed by proteolysis studies; Cook RA; Evidence is presented for the existence of altered ligand-stabilized conformational states of the NAD-specific malic enzyme (L-malate:NAD+ oxidoreductase (oxaloacetate-decarboxylating), EC 1.1.1.38), of Escherichia coli in the presence of Mg2+ and Mn2+, as identified by their susceptibilities to proteolysis . The rate of tryptic digestion of the enzyme is significantly decreased in the Mg2+-form of the enzyme when the product, NADH, or the allosteric effectors, coenzyme A and aspartate, are present in the digestion mixture . In contrast, little difference in the rate of tryptic digestion is observed in the degree of protection of the enzyme by the two metal cofactors, either alone, or in the presence of the substrates, malate and NAD . The results are consistent with the previously proposed hypothesis of Milne and Cook (Biochemistry 18, (1979) 3604-3610) that Mg2+ and Mn2+ stabilize two distinct conformational states of the enzyme . The results are discussed in relation to the altered kinetic response of the enzyme to substrates and effectors in the presence of the two metal cofactors. FEBS Lett, 1983 Dec 12, 164(2), 241 - 3 Nitrate, but not silver, ions induce spectral changes in Escherichia coli cytochrome d; Hubbard JA et al.; The absorbance maximum (630 nm) of reduced cytochrome d in Escherichia coli membrane particles was diminished by 160 microM AgNO3 or NaNO3 and accompanied by the formation of a species with an absorption maximum at 640-645 nm . Nitrite, trioxodinitrate and nitric oxide elicited qualitatively similar, but faster, changes in the spectrum of cytochrome d, suggesting that formation of a nitrosyl complex may be involved in all cases . In direct contrast to an earlier report, silver ions (160 microM) were without effect on the alpha-bands of reduced cytochromes d, b or a 1. Nucleic Acids Res, 1983 Dec 10, 11(23), 8407 - 14 The nucleotide sequences of tRNASer (GCU) and tRNAGln (UUG) genes from tobacco chloroplasts; Deno H et al.; The nucleotide sequence of tobacco chloroplast genes for tRNASer (GCU) and tRNAGln (UUG) have been determined . These tRNA genes are encoded on the same DNA strand and separated by 1144 bp . Two open reading frames of 52 codons and 98 codons have been found in this spacer region . The tRNASer (GCU) and tRNAGln (UUG) deduced from the DNA sequences show 67% and 76% sequence homologies with E . coli tRNASer (GCU) and tRNAGln (UUG), respectively. Nucleic Acids Res, 1983 Dec 10, 11(23), 8509 - 18 The DNA sequence of argI from Escherichia coli K12; Bencini DA et al.; The argI gene from E . coli K12 has been sequenced . It contains an open reading frame of 1002 bases which encodes a polypeptide of 334 amino acids . Three such polypeptides are required to form the functional catalytic trimer (c3) of ornithine transcarbamoylase (OTCase-1, EC 2.1.3.3) . The molecular mass of the mature trimer deduced from the amino acid sequence is 114,465 daltons . An altered form of argI was produced when a 1.6 kilobase DdeI fragment was subcloned into the HincII site of plasmid pUC8 extending the open reading frame an additional 20 nucleotides . It has been previously reported that the amino-terminal region of the respective polypeptides of argI, argF, and pyrB of E . coli possessed significant homology . In contrast, the homologous promoter/operator regions of argI and argF did not appear to share any homologies with pyrB . However, a closer scrutiny of the nucleotide sequence immediately preceding the pyrBI attenuator revealed a remarkable similarity to the argI and argF control region. Nucleic Acids Res, 1983 Dec 10, 11(23), 8283 - 6 supG and supL in Escherichia coli code for mutant lysine tRNAs+; Prather NE et al.; We have determined the nucleotide sequences of lysine tRNAs isolated from strains containing one or the other of two Escherichia coli ochre suppressors, supG and supL . Each strain, besides producing wild-type lysine tRNA, has a mutant lysine tRNA species that apparently can read the polypeptide chain termination codons UAA and UAG . The mutant tRNAs from supG and supL strains are identical . In each case the suppressor tRNA has an A36 for U36 nucleotide substitution . Furthermore, the hypermodified nucleoside at position 37 has been changed from t6A to ms2i6A. J Biol Chem, 1983 Dec 10, 258(23), 14592 - 8 Ribosomal protein L7/L12 cross-links to proteins in separate regions of the 50 S ribosomal subunit of Escherichia coli; Traut RR et al.; The 50 S ribosomal subunits from Escherichia coli were modified by reaction with 2-iminothiolane under conditions in which 65 sulfhydryl groups, about 2/protein, were added per subunit . Earlier work showed that protein L7/L12 was modified more extensively than the average but that nearly all 50 S proteins contained sulfhydryl groups . Mild oxidation led to the formation of disulfide protein-protein cross-links . These were fractionated by urea gel electrophoresis and then analyzed by diagonal gel electrophoresis . Cross-linked complexes containing two, three, and possibly four copies of L7/L12 were evident . Cross-links between L7/L12 and other ribosomal proteins were also formed . These proteins were identified as L5, L6, L10, L11, and, in lower yield, L9, L14, and L17 . The yields of cross-links to L5, L6, L10, and L11 were comparable to the most abundant cross-links formed . Similar experiments were performed with 70 S ribosomes . Protein L7/L12 in 70 S ribosomes was cross-linked to proteins L6, L10, and L11 . The strong L7/L12-L5 cross-link found in 50 S subunits was absent in 70 S ribosomes . No cross-links between 30 S proteins and L7/L12 were observed. J Biol Chem, 1983 Dec 10, 258(23), 14245 - 52 Glucosamine-derived phospholipids in Escherichia coli . Structure and chemical modification of a triacyl glucosamine 1-phosphate found in a phosphatidylglycerol-deficient mutant; Takayama K et al.; Certain Escherichia coli mutants defective in phosphatidylglycerol biosynthesis accumulate novel glucosamine-derived phospholipids . We previously demonstrated that the simplest of these substance (lipid X) is a diacylglucosamine 1-phosphate bearing beta-hydroxymyristoyl groups at positions 2 and 3 (Takayama, K., Qureshi, N., Mascagni, P., Nashed, M . A., Anderson, L., and Raetz, C . R . H . (1983) J . Biol . Chem . 258, 7379-7385) . We now report the structural characterization of a triacylglucosamine 1-phosphate (designated lipid Y) that is also found in these mutants . Hydrolyzates of Y contain 2 mol of beta-hydroxymyristate and 1 mol of palmitate/mol of glucosamine . In the lipid, one of the beta-hydroxymyristates is amide-linked at position 2, while the two other fatty acyl groups are ester-linked . Fast atom bombardment mass spectrometry is used to confirm that Y is a monosaccharide derivative and that the molecular weight of Y as the free acid (C50H96NO13P) is 950.29 . Analysis of Y by proton NMR spectroscopy at 200 MHz reveals that the anomeric configuration is alpha . Further, one of the esterified fatty acid residues is attached to the 3 OH of the sugar, while the second is linked to an OH moiety of a hydroxymyristate . The 4 and 6 OH groups of the sugar are unsubstituted, as in E . coli lipid X . To establish the precise location of each esterified fatty acyl residue, we subjected Y to a very mild alkaline hydrolysis in the presence of triethylamine . This resulted in the selective removal of a single hydroxymyristoyl group . The triethylamine-treated derivative (lipid Y) has a molecular weight of 723 . NMR spectroscopy of Y shows that the 3 OH of the sugar is no longer substituted, while the beta OH of the remaining amide-linked hydroxymyristate is still esterified with palmitate . On the basis of these findings, we propose that lipid Y has the same fundamental structure as lipid X, except for the additional presence of a palmitoyl moiety on the N-linked hydroxymyristate . Presumably, lipid Y is synthesized from X by a selective acylation reaction. J Biol Chem, 1983 Dec 10, 258(23), 14200 - 5 The elongation factor Tu from Escherichia coli, aminoacyl-tRNA, and guanosine tetraphosphate form a ternary complex which is bound by programmed ribosomes; Pingoud A et al.; The interaction of the Escherichia coli elongation factor Tu guanosine tetraphosphate complex (EF-Tu ppGpp) with aminoacyl-tRNAs(aa-tRNA) was reinvestigated by gel filtration and hydrolysis protection experiments . These experiments show that EF-Tu X ppGpp like EF-Tu X GDP (Pingoud, A., Block, W., Wittinghofer, A., Wolf, H . & Fischer, E . (1982) J . Biol . Chem . 257, 11261-11267) forms a fairly stable complex with Phe-tRNAPhe, KAss being 0.6 X 10(5) M-1 at 25 degrees C . The binding of the EF-Tu X ppGpp X aa-tRNA complex to programmed ribosomes was investigated by a centrifugation technique . It is shown that this complex is bound codon-specific with KAss = 3 X 10(7) M-1 at 0 degrees C and that it stimulates peptidyl transfer . A numerical estimation of the intracellular concentration of EF-Tu X GTP X aa-tRNA and EF-Tu X ppGpp X aa-tRNA during normal growth and under the stringent response indicates that ppGpp accumulation does affect the EF-Tu X GTP X aa-tRNA concentration but does not lead to major depletion of this pool . Furthermore, due to the higher affinity of EF-Tu X GTP to aa-tRNA and of the ternary complex EF-Tu X GTP X aa-tRNA to the ribosome, EF-Tu X ppGpp X aa-tRNA binding to the ribosome is not significant . According to our measurements and calculations, therefore, a direct participation of EF-Tu in slowing down the rate of protein biosynthesis and improving its accuracy during amino acid starvation is not obvious. J Biol Chem, 1983 Dec 10, 258(23), 14116 - 9 Thiophosphorylation as a probe for subunit interactions in Escherichia coli succinyl coenzyme A synthetase . Further evidence for catalytic cooperativity and substrate synergism; Wolodko WT et al.; Succinyl-CoA synthetase has an (alpha beta)2 subunit structure and shows half-of-the-sites reactivity with respect to the formation of the phosphohistidyl residues that acts as a catalytic intermediate . Adenosine 5'-O-(3-thio)triphosphate has been found to be a substrate, but the overall maximum velocity is 3 orders of magnitude lower than that seen with ATP . Moreover, steps of the reaction involving thiophosphoryl transfer are much slower than the corresponding phosphoryl transfers . These properties of adenosine 5'-O-(3-thio)triphosphate as a substrate have been exploited to test the concept of alternating sites catalytic cooperativity proposed earlier as a rationale for the subunit structure of succinyl-CoA synthetase . As predicted by this model for catalysis, the rate of discharge of thiophosphate from the enzyme in the presence of succinate and CoA is stimulated by ATP . Neither of two nonhydrolyzable analogs of ATP has an equivalent effect . The results indicate that the transfer of the thiophosphoryl group from the enzyme to succinate at one active site is not favored until the neighboring active site is phosphorylated by ATP, with accompanying reciprocal changes in the conformations of the two halves of the enzyme molecule. J Biol Chem, 1983 Dec 10, 258(23), 14073 - 5 An unpaired 3' terminus stimulates recA protein-promoted DNA strand exchange; Soltis DA et al.; In the presence of 100 mM NaCl, the efficient exchange of strands between a circular single strand and an homologous DNA duplex promoted by the recA and single-stranded DNA binding proteins of Escherichia coli requires an unpaired 3' terminus . Of the duplex DNAs tested, only those with 4 unpaired bases at the 3' termini are effective . Without added NaCl, strand exchange proceeds efficiently with all duplex DNA termini examined including a nicked circular duplex . Thus, at approximately physiological salt concentrations, factors in addition to the recA and single-stranded DNA binding proteins are needed to promote efficient strand exchange . One such factor may be a DNA helicase(s). Nucleic Acids Res, 1983 Dec 10, 11(23), 8149 - 65 Telomere conversion in trypanosomes; De Lange T et al.; Activation of the gene coding for variant surface glycoprotein (VSG) 118 in Trypanosoma brucei proceeds via a duplicative transposition to a telomeric expression site . The resulting active expression-linked extra copy (ELC) is usually flanked by DNA that lacks sites for most restriction enzymes and that is thought to interfere with the cloning of the ELC as recombinant DNA in Escherichia coli . We have circumvented this problem by cloning an aberrant 118 ELC gene, flanked at the 3'-side by at least 1 kb DNA, that contains restriction enzyme sites . Our analysis shows that this DNA and the 3'-end of the 118 ELC gene are derived from another VSG gene (1.1006) that is permanently located at a telomeric position . We propose that the 3'-end of the 1.1006 gene and (all of) its 3' flanking sequence moved to the expression site by a telomere conversion . Such a telomere conversion can also account for the appearance of an extra copy of the 1.1006 gene detected in a sub-population of our trypanosome strain. J Biol Chem, 1983 Dec 10, 258(23), 14599 - 609 Cross-linking and labeling of the Escherichia coli F1F0-ATP synthase reveal a compact hydrophilic portion of F0 close to an F1 catalytic subunit; Aris JP et al.; The subunit arrangement of the F0 sector of the Escherichia coli ATP synthase is examined using hydrophilic and hydrophobic (cleavable) cross-linking reagents and the water-soluble labeling reagent {35S} diazoniumbenzenesulfonate ( {35S}DABS) . Cross-linking is performed on purified ATP synthase and inverted minicell membranes . ATP synthase incorporated into liposomes is labeled with {35S}DABS . Three cross-linked products involving the F0 subunits (a, b, and c) are observed with the purified ATP synthase in solution: a-b, b2, and c2 dimers . A cross-link between the F0 and F1 is detected and occurs between the a and beta subunits . A cross-linker independent association between the b and beta subunits is also evident, suggesting that the two subunits are close enough to form a disulfide bridge . A cross-linking reagent stable to reducing agents produces a b-beta dimer, as detected by immunoblotting with anti-beta serum . The c subunit does not cross-link with any F1 polypeptide . Minicell membranes containing ATP synthase polypeptides radioactively labeled in vivo similarly show b2 and c2 dimers after cross-linking . {35S}DABS labels the a and b, but not c, subunits, showing that the a and b, but not c, subunits possess hydrophilic domains . Thus, certain domains of subunits a and b extend from the membrane and are in close proximity to one another and the F1 catalytic subunit beta. J Biol Chem, 1983 Dec 10, 258(23), 14550 - 5 Topology, organization, and function of the psi subunit in the F0 sector of the H+-ATPase of Escherichia coli; Hermolin J et al.; The F1F0 H+-ATPase in membranes of Escherichia coli was amplified by heat induction of a lysogenic lambda-unc+ transducing phage . Inverted membrane vesicles were stripped of the F1 sector of the ATPase complex by washing with EDTA . The stripped membranes were treated with dithiobis(succimidylpropionate) to cross-link subunits of the F0 sector of the ATPase complex . After electrophoresis under nonreducing conditions in one dimension, cross-linked subunits were identified by off-diagonal electrophoresis in a second dimension following cleavage of the cross-linked products with beta-mercaptoethanol . A psi-psi dimer was the major cross-linked product identified . In addition, a chi-psi product and chi-psi2 product were identified . These results support the proposed chi-psi2 stoichiometry of subunits in F0 . When the F1-stripped membranes were treated with trypsin, the psi subunit was rapidly degraded, whereas psi was protected from degradation when F1 was bound to the membrane . Trypsin-treated, stripped membranes, lacking an intact psi subunit, did not bind the F1 portion of the ATPase with high affinity . However, these trypsin-treated stripped membranes remained as permeable to protons as untreated stripped membranes, and the H+ conductivity was blocked by dicyclohexylcarbodiimide . These results indicate that the portion of the psi subunit exposed on the cytoplasmic face of the inner membrane is involved in the binding of the F1 portion of the ATPase, but is not necessary for H+ conduction mediated by the F0 sector of the complex. J Biol Chem, 1983 Dec 10, 258(23), 14478 - 84 Plastid translation in organello and in vitro during light-induced development in Euglena; Miller ME et al.; Plastids were isolated from dark-grown Euglena gracilis, from cells exposed to light for 3 h, and from normal, light-grown cells . Plastid protein synthesis was then measured either in organello, by incorporation of {35S}Met into isolated plastids, or in vitro, by programming wheat germ, reticulocyte, or Escherichia coli translation systems with RNA isolated from the plastids . Specific and total rates of protein synthesis in organello increased during light-induced development about 100-fold . In contrast, specific mRNA activity of plastid RNA increased no more than 3-fold, and the estimated total mRNA activity of plastids increased less than 10-fold . Protein synthesis in plastids appears therefore to be subject to strong regulation at a level other than that of transcription superimposed on weak transcriptional regulation . The synthesis of the large subunit of ribulose-bisphosphate carboxylase in particular appears to be under translational control . There is also qualitative evidence for regulation . Most plastid mRNAs, as indicated by translation in vitro, electrophoresis, and fluorography of their translation products, increase with light-induced development as a coordinate set . A second or proplastid set can be detected in RNAs from immature plastids, but not from mature chloroplasts . A few mRNAs appear only later in development, corresponding to a delayed set . Most translation products measured in organello also appear to belong to the coordinate set, a few to the proplastid and delayed sets, and a further few to a transient set, which appears only early in development. J Biol Chem, 1983 Dec 10, 258(23), 14632 - 7 Regulation of tyrosine hydroxylase mRNA by glucocorticoid and cyclic AMP in a rat pheochromocytoma cell line . Isolation of a cDNA clone for tyrosine hydroxylase mRNA; Lewis EJ et al.; Treatment of a subclone of the PC12 pheochromocytoma cell line, PC8b, with either dexamethasone or 8-bromo cyclic AMP resulted in increased translational activity of tyrosine hydroxylase mRNA (mRNATH) . Poly(A+)-containing RNA from cells treated with both inducers was used to construct a cDNA library . Double-stranded cDNA was inserted into the PstI site of pBR322 using GC tailing, and plasmids were used to transform Escherichia coli HB101 . Colonies containing plasmids with inserted sequences were initially screened by DNA dot hybridization, and those positive colonies were then screened by hybrid selected translation . One plasmid, pTH.4, was identified as containing a 400-base pair sequence complementary to mRNATH . Nick-translated pTH.4 DNA was used to identify mRNATH as containing approximately 1800 nucleotides by Northern blot analysis . PC8b cells treated with either dexamethasone or 8-bromo cyclic AMP yielded greater mRNATH hybridization on Northern blot analysis and accumulated higher molecular weight tyrosine hydroxylase RNA species . Following treatment of cells with inducers, the temporal increase in tyrosine hydroxylase enzyme activity was associated in all cases with an increase in the translational activity and relative amount of mRNATH, and the fold increase in the latter two parameters was equal to or greater than the increase in enzyme activity. Eur J Pharmacol, 1983 Dec 9, 96(1-2), 11 - 9 Antisecretory effects of berberine with morphine, clonidine, L-phenylephrine, yohimbine or neostigmine in pig jejunum; Zhu B et al.; The effects of berberine alone or in combination with morphine, clonidine, L-phenylephrine or yohimbine were compared in Escherichia coli heat-stable enterotoxin (ST)-exposed ligated jejunal loops in 2 week old pigs . In addition, net water and electrolyte fluxes in normal jejunal loops were measured in the presence of neostigmine, morphine, clonidine, L-phenylephrine or yohimbine alone or in combination with berberine . Berberine, morphine, clonidine and L-phenylephrine each reduced the net secretion of water and electrolytes induced by ST (P less than 0.05) . A significant enhancement of antisecretory effect was observed only with the combination of berberine and L-phenylephrine . Yohimbine or neostigmine augmented the net loss of water and electrolytes produced by ST . Yohimbine did not block the antisecretory action of berberine . In normal jejunum, there was no significant difference in water and ion absorption between adrenergic or opiate agonists alone and their combination with berberine . Neostigmine reversed absorption to net secretion in normal jejunum and this effect was significantly reduced by berberine . The anti-secretory action of berberine appears similar to that of alpha 2-adrenergic agonists, opiates, and anticholinergic agents. Biochemistry, 1983 Dec 6, 22(25), 5897 - 902 Beta-hydroxydecanoyl thio ester dehydrase does not catalyze a rate-limiting step in Escherichia coli unsaturated fatty acid synthesis; Clark DP et al.; The intracellular level of beta-hydroxydecanoyl thio ester dehydrase, the product of the fabA gene of Escherichia coli, was increased by isolation of a putative promotor mutant (termed fabAup) or by molecular cloning of the wild-type fabA gene into plasmid pBR322 . The fabAup and plasmid-carrying strains overproduced dehydrase by about 15- and 10-fold, respectively . The phospholipids of all strains that overproduced the dehydrase contained significantly higher levels of saturated fatty acids than isogenic strains producing a normal level of dehydrase . No increased levels of unsaturated fatty acids were observed . This result indicates that, although the dehydrase is required for unsaturated fatty acid synthesis, the level of dehydrase activity in wild-type cells does not limit the rate of unsaturated fatty acid synthesis . The introduction of a plasmid carrying the structural gene for beta-ketoacyl acyl carrier protein synthase I into a fabAup strain overcame the effect of dehydrase overproduction on fatty acid composition. Biochemistry, 1983 Dec 6, 22(25), 5754 - 60 Stereospecificity and requirements for activity of the respiratory NADH dehydrogenase of Escherichia coli; Campbell HD et al.; The respiratory NADH dehydrogenase of Escherichia coli has been further amplified in vivo by genetic methods . The enzyme, a single polypeptide of Mr 47 200 of known amino acid sequence {Young, I . G., Rogers, B . L., Campbell, H . D., Jaworowski, A., & Shaw, D . C . (1981) Eur . J . Biochem . 116, 165-170}, constitutes 10-15% of the total protein in the amplified membranes . In situ in the membrane, the enzyme contains 1 mol of FAD/mol of subunit and has a specific NADH:ubiquinone-1 oxidoreductase activity of approximately 1100-1200 units mg-1 at 30 degrees C, pH 7.5 . The purified enzyme contains phospholipid, which remains closely associated with it during gel filtration on Sephacryl S-300 in the presence of 0.1% (w/v) cholate at low ionic strength . Under these conditions the enzyme is extensively aggregated (apparent Mr greater than 10(6} . This procedure yielded enzyme with a specific activity of 980 units mg-1, similar to the value observed in the membrane . This preparation contained less than 0.1 mol of Fe/mol of enzyme, confirming that Fe is not involved in reduction of ubiquinone 1 catalyzed by the enzyme . Neutron activation analysis of purified enzyme has demonstrated the absence of 35 trace elements including Se, Zn, Mn, Co, W, Cu, and Fe . The enzyme polypeptide, prepared completely free of phospholipid, FAD, and ubiquinone by gel filtration in the presence of sodium dodecyl sulfate, has been reactivated . The results show that the only components necessary for catalysis of ubiquinone-1 reduction by NADH in this system are the enzyme polypeptide, FAD, and phospholipid.(ABSTRACT TRUNCATED AT 250 WORDS) J Mol Biol, 1983 Dec 5, 171(2), 207 - 15 Crystallization of a ribonuclease-resistant fragment of Escherichia coli 5 S ribosomal RNA and its complex with protein L25; Abdel-Meguid SS et al.; A ribonuclease-resistant fragment of Escherichia coli 5 S ribosomal RNA has been crystallized . The space group is P6(1)22 or P6(5)22, with a = 59.5 A and C = 268 A . The crystals contain one molecule per asymmetric unit, and show diffraction to 4.0 A resolution . Also, a complex of this fragment with L25 ribosomal protein has been crystallized in the same space group, but with a = 119 A, c = 250 A and four molecules per asymmetric unit. Nippon Geka Gakkai Zasshi, 1983 Dec, 84(12), 1213 - 9 {Studies on metabolism of fat emulsion in endotoxemic dogs}; Mori H; Changes of fat metabolism in endotoxemic dogs were studied by means of intravenous fat tolerance test . Twelve dogs subjected to the present studies were divided into two groups; peritonitis was produced in one group (6 dogs) and 1.5 mg/kg of E . coli lipopolysaccharide was injected intravenously in another groups (6 dogs) . After production of peritonitis, Limulus test was positive in all of the six dogs, however there were no significant changes in blood sugar levels, lactate/pyruvate ratio, and elimination rate of fat emulsion from the venous blood (K-value) after the induction of endotoxemia by peritonitis . On the other hand, a significant fall of the blood sugar levels and an increase of lactate/pyruvate ratio were found after the injection of endotoxin . A significant fall of K-value (from 0.0133 +/- 0.0056 to 0.0069 to 0.0024) was also observed in these six dogs . Results of the present studies suggest that the metabolism of fat emulsion is significantly affected by the intravenous injection of endotoxin, but not by the production of peritonitis if there is no disturbance of tissue perfusion. Bull Soc Pathol Exot Filiales, 1983 Dec, 76(5 Pt 2), 818 - 24 {Incidence of intestinal parasitosis among the Batwa and Hutu pygmy tribes of Rwanda}; Scaglia M et al.; The AA have carried out an epidemiological investigation on the incidence of intestinal parasitosis in groups of populations belonging to the pygmoid tribes Batwa and Hutu living in the Northern and Southern regions of Rwanda (East Africa) . Positivity for intestinal parasites (protozoa and/or helminths) reached 100% in the 309 subjects examined, no significant difference in prevalence being observed between the Northern and Southern groups . Among the protozoa, E . coli and E . histolytica were most commonly found, while trichocephalosis and ascaridiasis were the most frequently encountered helminthiasis . The occasional finding of Strongyloides fulleborni infestation should also be pointed out. Acta Physiol Scand, 1983 Dec, 119(4), 373 - 9 Sympathetico-adrenergic influences on the small intestinal vascular reactions in experimental septic shock; Falk A et al.; The objective of this study was to explore the vascular reactions in the small intestine and the possible role of sympathetico-adrenergic influences . The experiments were performed on 23 cats . The small intestinal blood flow was estimated using a drop counting technique . In one series of cats (n = 7) the small intestine and the adrenal glands had intact vascular and nervous supply (I), in another series (n = 7) the small intestine was sympathetically denervated and the adrenal vessels ligated (II), and in a third series (n = 9) the small intestine was innervated but the adrenal vessels ligated (III) . The septic state was induced by i.v . infusion of live E . coli bacteria for two hours . The small intestinal blood flow decreased and intestinal blood flow resistance increased in all series within 3 min upon bacterial infusion . The intestinal vasoconstriction was maintained in cats with intestinal denervation as well as in cats with the adrenal vessels ligated, favouring that other humoral factors than catecholamines are involved . During the later phase of bacteremic shock the intestinal blood flow remained in the preseptic range in the series with a denervated small intestine and ligated adrenals as well as in intact cats, but declined gradually and significantly in cats with adrenal ligation only (III) . This pattern of reactions favours a local rather than a remote sympathetico-adrenal influence. Anal Biochem, 1983 Dec, 135(2), 318 - 25 Improved conditions for activity gel analysis of DNA polymerase catalytic polypeptides; Karawya E et al.; In a study of mouse DNA polymerase catalytic polypeptides using activity gel analysis, it was found that the sensitivity of detection of purified enzymes is markedly increased by addition of a heterogeneous mixture of proteins to the enzyme sample prior to electrophoresis (Karawya E., and Wilson, S.H . (1982) J . Biol . Chem . 257, 13,129-13,134) . This modification and the use of a micromolar level of {32P}dNTP substrate are the basis of an improved activity gel assay for DNA polymerase catalytic polypeptides . This modified assay is several orders of magnitude more sensitive than the original procedure (Spanos, A., Sedgwick, S.G., Yarranton, G.T., Hubscher, U., and Banks, G.R . (1981) Nucl . Acids Res . 9, 1825-1839), and it enables measurement of two reference enzymes, calf beta-polymerase and Escherichia coli DNA polymerase I large fragment, in the picogram range . Further, it was found that it is essential to survey different lots of sodium dodecyl sulfate to identify those which enable high enzyme activity signals after renaturation. Biol Reprod, 1983 Dec, 29(5), 1241 - 53 Rat sperm are mechanically immobilized in the caudal epididymis by "immobilin," a high molecular weight glycoprotein; Usselman MC et al.; In many mammals, sperm are immotile while stored in the caudal epididymis and do not become motile until ejaculation . We report here our investigation of the mechanism that initiates motility in mature rat epididymal sperm . We found that an external "activator" is not required to initiate rat sperm motility since immotile sperm started to swim immediately when exposed to solutions that contributed only osmotic support . Instead, we found that epididymal rat sperm are kept fully immobilized by a high molecular weight glycoprotein, "immobilin," that we have isolated from rat cauda epididymal (CE) fluid . Our results strongly support the hypothesis that immobilin inhibits sperm motility mechanically simply by creating a highly viscoelastic environment: 1) rat CE fluid inhibited the motility of such disparate cells as rat sperm, E . coli . and rabbit sperm (which are fully motile in rabbit CE fluid), 2) the degree to which a variety of enzymatic treatments or slight dilution of the CE fluid initiated sperm motility depended only on the degree to which the treatment reduced the viscoelastic drag of the fluid, and 3) centrifugation of CE fluid simultaneously copurified the component of the fluid that immobilizes the sperm, the component that renders the fluid viscoelastic, and the glycoprotein immobilin. Biomed Mass Spectrom, 1983 Dec, 10(12), 660 - 4 An evaluation of the in vivo metabolism of cystine in Escherichia coli using stable isotopes; White RH; The incorporation of deuterated serine into cysteine during the metabolism of cystine by Escherichia coli was studied in order to determine the extent to which the carbon-sulfur bond(s) of the cystine is cleaved . The results indicate that the major route (approximately 80%) for cystine metabolism consists of a reductive cleavage of the cystine disulfide bond to form cysteine . Evidence is presented which shows that a portion of the remaining cystine is broken down by a pathway(s) which results in cleavage of the carbon-sulfur bond of the cystine . This pathway would be the same as that expected for the beta-elimination of pyruvate from cystine catalysed by the enzyme beta-cystathionase . In addition, a small portion of the resulting cysteine is shown to undergo a reversible dissociation to serine and hydrogen sulfide . Evidence is presented which shows that this dissociation is caused by the enzyme cysteine synthetase {O-acetyl-L-serine acetate-lyase (adding H2S)}. Cell, 1983 Dec, 35(2 Pt 1), 403 - 10 New heat shock puffs and beta-galactosidase activity resulting from transformation of Drosophila with an hsp70-lacZ hybrid gene; Lis JT et al.; A hybrid gene that consists of the Drosophila heat shock gene, hsp70, fused to the E . coli beta-galactosidase gene has been introduced into the Drosophila germline by the P element microinjection method . This hybrid includes 194 bp of sequence upstream of the start of the hsp70 transcript . Three strains of transformed flies were isolated and characterized by DNA blotting experiments and by in situ hybridization to polytene chromosomes . Strain Bg61 has a single insert of the hybrid gene at the tip of chromosome 3L, site 61A, and the insert consists of a structure that is consistent with P-element-mediated transposition . Strain Bg9,61 has inserts at both 61A and 9E, while Bg64 has a single insert at 64D . Heat shock induces the formation of a large chromosomal puff at all three sites . These puffs appear and regress with kinetics indistinguishable from the puffing of the heat shock locus, 87C, from which the hsp70 gene, used in these studies, was isolated . The beta-galactosidase activity in the transformants is inducible by heat shock and shows a widespread distribution throughout the tissues of larvae and adults. J Exp Med, 1983 Dec 1, 158(6), 2159 - 64 Cloned mouse interferon-gamma inhibits the growth of Rickettsia prowazekii in cultured mouse fibroblasts; Turco J et al.; The effect of treating cultured mouse fibroblasts (L929 cells) with cloned mouse interferon-gamma on the growth of Rickettsia prowazekii within the fibroblasts was studied . Within 48 h after infection, rickettsiae were cleared from a substantial proportion of the initially infected cells and rickettsial growth was inhibited in those cells that remained infected, when L929 cells were treated with cloned mouse interferon-gamma both before and after infection . When L929 cells were treated with cloned mouse interferon-gamma either only before or only after infection with rickettsiae, rickettsial growth was markedly inhibited but rickettsiae were not cleared from many cells . Addition of cycloheximide to L929 cells markedly suppressed the antirickettsial activity of the interferon, and cloned mouse interferon-gamma did not induce antirickettsial activity in human foreskin fibroblasts . The antirickettsial effects of cloned mouse interferon-gamma were similar to those induced by crude mouse lymphokines prepared from concanavalin A-stimulated mouse spleen cells . Equivalent amounts (units) of cloned mouse interferon-gamma produced by Chinese hamster ovary cells or by Escherichia coli caused equivalent inhibition of rickettsial growth in mouse fibroblasts . However, at high concentrations of interferon-gamma, treatment of rickettsia-infected fibroblasts with equivalent amounts (units) of interferon-gamma, as crude mouse lymphokines or cloned mouse interferon-gamma, resulted in slightly greater inhibition of rickettsial growth by the crude lymphokines . Most of the antirickettsial activity of crude mouse lymphokines can be explained by the interferon-gamma that is present in these preparations . Interferon-gamma, by virtue of its ability to inhibit rickettsial growth and effect the clearance of rickettsia from nonprofessional phagocytes, may play a crucial role in the elimination of rickettsiae from the infected host. J Immunol, 1983 Dec, 131(6), 2821 - 6 Activation of human monocyte cytotoxicity by natural and recombinant immune interferon; Le J et al.; Human T cell hybridomas were established by fusion of SH9 cells, the 6-thioguanine-resistant mutant line of human T lymphoma Hut 102-B2, with concanavalin A-stimulated human peripheral blood lymphocytes . Hybridoma line L38 produced a macrophage activating factor (MAF) with the ability to activate human peripheral blood monocytes to show enhanced cytotoxicity against human colon adenocarcinoma HT-29 cells in a 72-hr 125iododeoxyuridine-release assay . The L38 line was then cloned by the limiting dilution technique and two sublines, L38B and L38D, were found to produce high levels of MAF constitutively . Interferon activity was also detected in L38B and L38D supernatants . When interferon activity was neutralized with specific antiserum to purified human immune interferon (IFN-gamma), MAF activity was abrogated . To confirm that the MAF activity is indeed due to IFN-gamma, IFN-gamma was purified from the culture supernatant of another human T cell hybridoma, L265K2, a cell line known to produce high levels of IFN-gamma . Two highly purified IFN-gamma fractions with m.w . of 20,000 and 25,000, respectively, were obtained by NaDodSO4/polyacrylamide gel electrophoresis (SDS-PAGE) . Similar fractions were obtained from IFN-gamma derived from human peripheral blood lymphocyte (PBL) cultures induced with 12-0-tetradecanoylphorbol-13-acetate (TPA) and phytohemagglutinin (PHA) . In comparison, Escherichia coli-derived recombinant human IFN-gamma separated by SDS-PAGE yielded two major active fractions with m.w . of 17,000 and 34,000 . With all three types of preparations, a close correlation was found between the presence of IFN-gamma activity demonstrable in an antiviral assay and MAF activity in individual fractions . Substantial quantitative differences were observed in the ability of various human IFN to activate monocytes . Although no MAF activity was detected with IFN-alpha and IFN-beta at concentrations up to 200 U/ml, both natural and recombinant IFN-gamma showed marked MAF activity at concentrations as low as 0.3 to 1 U/ml. J Bacteriol, 1983 Dec, 156(3), 1198 - 203 Regulatory pattern of the Escherichia coli lysA gene: expression of chromosomal lysA-lacZ fusions; Stragier P et al.; The regulation of lysA which encodes the last enzyme for lysine biosynthesis in Escherichia coli, diaminopimelic acid-decarboxylase, was studied by using lysA-lacZ fusions . Our results indicate an absolute requirement for the LysR product for its activation, LysR protein present in a limiting amount which can be titrated by a multicopy plasmid carrying its target site and a negative regulatory role for the LysA protein itself which decreases lysA-lacZ expression 30-fold. J Biomol Struct Dyn, 1983 Dec, 1(3), 647 - 56 Use of psoralen monoadducts to compare the structure of 16S rRNA in active and inactive 30S ribosomal subunits; Chu YG et al.; E . coli 30S ribosomes in the inactive conformation were irradiated at 390 nm in the presence of 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT) . This produces monoadducts in which AMT is attached to only one strand of an RNA duplex region . After unbound AMT was removed, some ribosomes were activated and then subjected to 360 nm irradiation; others were reirradiated without activation . Electron microscopic examination of 16S rRNA extracted from these two samples showed covalent rRNA loops indicative of rRNA crosslinks . The general pattern of loops closely matched that seen previously after direct psoralen crosslinking of 30S particles . However, the frequency of occurrence of one major class of loops formed by crosslinks between residues near position 500 and the 3' end was substantially lower for the activated samples, implying that the structure of the 16S rRNA in active and inactive 30S particles is different. J Biomol Struct Dyn, 1983 Dec, 1(3), 593 - 609 The in-vivo occurrence of Z DNA; Haniford DB et al.; The energetics of the B-Z transition of two different types of cloned alternating purine/pyrimidine DNA sequences have been analysed by a two dimensional electrophoretic technique . Since the transition between right handed and left handed forms of these polymers is detected by alterations of electrophoretic mobilities of topoisomers of the plasmid DNA molecules, the method is not dependent on Z-DNA binding ligands . The measurements reflect intrinsic properties of the DNA unperturbed by the free energy of binding such a ligand . Direct evidence from the analysis of topoisomer distributions is presented which shows that d(GC)n.d(GC)n sequence elements within an E . coli plasmid will adopt a Z conformation in-vivo under conditions of blocked protein synthesis . Evidence for the in-vivo occurrence of Z-DNA was not detected in plasmid DNA isolated from bacterial cells growing in the absence of protein synthesis inhibitors . A model is proposed for a function for the B-Z transition in ensuring the correct pairing of homologous chromosomes during meiosis. Mol Biol Evol, 1983 Dec, 1(1), 67 - 83 Geographic components of linkage disequilibrium in natural populations of Escherichia coli; Whittam TS et al.; Geographic variation in the genetic structure of natural enteric populations of Escherichia coli was assessed at both the single-locus and dilocus levels from allozyme genotypes at 12 enzyme loci in 178 cell lines isolated from human hosts in Sweden, Iowa, and Tonga . Although there was significant heterogeneity in allele frequencies at six of the 12 loci, geographic variation accounted for only 2.0% of the total genetic diversity (HT = 0.518) . Ohta's D-statistics were used to partition the total variance of dilocus linkage disequilibrium into within-population and between-population components . The observed total variance in disequilibrium (0.0339), averaged over 66 locus-pairs, was significantly greater than would be expected (0.0103) if alleles were randomly associated in an unstructured total population; and both within-locality and between-locality components made substantial contributions to the total variance . Half the locus-pairs exhibited the specific dual relationship among components expected when random factors are generating disequilibrium, but 20% of the locus-pairs showed the opposite relationship, reflecting systematic allele associations . The magnitude of dilocus disequilibrium apparently is unrelated to the chromosomal distance between loci . This and other evidence indicates that substitutive recombination rates in natural populations are sufficiently low to permit indirect periodic selection to play a prominent role in generating multilocus genetic structure. Rev Esp Fisiol, 1983 Dec, 39(4), 357 - 62 Study of several factors affecting the agglutinating activity of K99-positive Escherichia coli strains; Ferreiros CM et al.; The effect of several factors on Escherichia coli K99-plasmid associated agglutination has been studied . The results obtained indicate that Escherichia coli 637 (K99+) mediated red blood cell agglutination is unspecific although the agglutination titres for several erythrocyte species are significantly different . The agglutination is highly stable (at least with sheep red blood cells) to changes in temperature (from 4 degrees C to 37 degrees C), to changes in pH (from 5 to 9) and to the presence or absence of several metallic cations . Treatment of sheep erythrocytes with certain proteolytic enzymes (papain and trypsin) results in a increment in their agglutinability . Also, the extraction of galactose after the removal of sialic acid residues from the oligosaccharides present on the erythrocyte membranes results in a great increment in their agglutinability . On the other hand, only thyroglobulin, mucin, fetuin, and the oligosaccharides extracted from the last two glycoproteins are able to inhibit K99-plasmid mediated sheep red blood cell agglutination. Proc Natl Acad Sci U S A, 1983 Dec, 80(24), 7380 - 4 Inducible repair of phosphotriesters in Escherichia coli; McCarthy JG et al.; Extracts from Escherichia coli cells induced for the adaptive response have been prepared that are capable of repairing O6-methylguanine, O4-methylthymine, and the phosphotriesters produced on the DNA backbone by alkylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) . The phosphotriesters are repaired by a methyltransferase distinct from the one that demethylates O6-methylguanine . We propose that this increased capacity to repair phosphotriesters accounts for much of the increased resistance to MNNG toxicity seen in cultures induced for the adaptive response. J Biochem (Tokyo), 1983 Dec, 94(6), 1789 - 95 Phosphoenolpyruvate carboxylase of Escherichia coli . Inhibition by various analogs and homologs of phosphoenolpyruvate; Izui K et al.; In an attempt to investigate the topography of the catalytic site of phosphoenolpyruvate (PEP) carboxylase {EC 4.1.1.31} of Escherichia coli, the inhibitor constants (Ki) for more than 20 compounds were determined with the reaction system containing dioxane, a non-physiological activator of the enzyme . The Ki values for the compounds lacking methylene-, carboxylate-, or phosphate groups were all more than 10-fold larger than the Km value for PEP, indicating the significant contribution of these groups to the binding of PEP with the enzyme . The Ki value for L-phospholactate (0.30 mM) was almost equal to the Km value for PEP (0.25 mM), whereas that for D-phospholactate (0.89 mM) was about 3-fold larger than the Km value . It was presumed that PEP binds with the enzyme on its si-side . Among 6 PEP homologs, the Ki values for phosphoenol alpha-ketobutyrate (0.024 mM) and phosphoenol alpha-ketovalerate (0.034 mM) were about one-tenth the Km value, indicating the presence of a hydrophobic pocket around the binding site of the methylene group of PEP, where the carboxylation reaction is supposed to occur . DL-Phosphomalate, a presumptive carboxylated substrate, was a weak inhibitor with a Ki value of 2.20 mM. Gene, 1983 Dec, 26(2-3), 165 - 70 Structural organization of the genes that encode two glutamate synthase subunits of Escherichia coli; Garciarrubio A et al.; Plasmid pRSP20, a recombinant plasmid isolated from the Clarke-Carbon Escherichia coli gene bank, contains the two genes coding for the subunits of glutamate synthase (GOGAT) . We have constructed several derivatives of pRSP20, and analyzed the direction of transcription and genetic organization of these genes . Unexpectedly, we have found that although they are tightly linked and are transcribed in the same direction, each of them has its own promoter. Gene, 1983 Dec, 26(1), 79 - 89 The control region of the F plasmid transfer operon: DNA sequence of the traJ and traY genes and characterisation of the traY leads to Z promoter; Fowler T et al.; The complete nucleotide sequence of the F plasmid transfer genes traJ and traY, together with the promoter-proximal region of the traA gene has been determined . The traJ reading frame has been confirmed by sequencing the traJ90 amber mutant allele . The predicted amino acid sequence of the TraJ protein shows that this outer-membrane protein lacks a signal sequence . The pattern of codon usage within the traJ gene is different from that of genes for abundant outer-membrane proteins and is closer to that of genes that are expressed at relatively low levels . We have located the traY leads to Z operon promoter by in vitro run-off transcription experiments and have developed in vivo assays for the activity of the promoter by fusing it to galactokinase and kanamycin-resistance genes. Prostaglandins Leukot Med, 1983 Dec, 12(4), 385 - 97 A pathophysiological role of endogenous prostacyclin in endotoxin induced increase in lung vascular permeability in dogs; Ikeda T et al.; Escherichia coli endotoxin (1 mg/kg) infusion over 30 min into anesthetized artificially ventilated dogs caused a biphasic response: an early phase of pulmonary hypertension and a late phase of increased lung vascular permeability . During an early phase, PG F2 alpha, Tx A2 (as Tx B2) and prostacyclin (as 6-keto-PG F1 alpha) concentrations increased in plasma or right duct lymph of dogs . During a late phase, the concentrations of PG F2 alpha and Tx A2 decreased to near the base-line values, while the concentration of prostacyclin remained elevated . Administrations of PG synthetase inhibitors 45 min prior to endotoxin inhibited the increase in concentration of prostacyclin following the infusion of endotoxin and potentiated the increase in lung vascular permeability at the beginning of the late phase . Continuous infusion of prostacyclin (20 ng/kg/min) starting one hour before endotoxin for 5 hour periods prevented the increase in lung vascular permeability induced by endotoxin . Based on these results, we could conclude that endogenous prostacyclin might play an important role in preserving cell integrity of lungs and counteract the deleterious effects of endotoxin. J Gen Microbiol, 1983 Dec, 129 ( Pt 12), 3671 - 7 The unsuitability of the uridine incorporation assay for the measurement of phagocytosis of Escherichia coli; Fazeli A et al.; The uridine incorporation technique for assaying phagocytosis is based on the fact that polymorphonuclear leucocytes are impermeable to labelled uridine, and therefore ingested bacteria inside phagocytic vacuoles will be unable to take it up . Extracellular bacteria, including those adherent to the phagocytic cell surface, can do so however . Differences in uptake between bacteria alone and in the presence of phagocytic cells can be used to measure ingestion . The present paper describes the application of this technique to Escherichia coli O-86 as the test organism . It appears that with this test species, the method is unsuccessful, because exposure of the non-ingested bacteria to some soluble product of the triggered polymorphonuclear leucocytes causes a large increase in their uridine uptake rates, over that of the control bacteria . The nature of the product responsible is unknown . It is unconnected with change in the pH of the medium, is heat stable, and is only produced by polymorphonuclear leucocytes which are actively phagocytosing . It may be that a release of phagolysosome contents is responsible. J Dairy Sci, 1983 Dec, 66(12), 2593 - 6 Comparison of teat dips with differing iodine concentrations in prevention of mastitis infection; Bray DR et al.; Six commercial dairy herds were used to test the relative efficacy of three concentrations of iodine as a teat dip over 12 mo . Concentrations were .1, .25, and 1% iodine with free iodine contents of 3.5, 4.0, and 1.0 ppm . Two concentrations were compared in each herd . The greatest number of new infections (71) occurred in approximately 120 cows whose teats were dipped with the 1% iodine dip, and the fewest (52) occurred in a similar number of cow quarters dipped in .1% iodine; however, differences were not significant . Clinical mastitis was highest in the 1% group . If all clinical mastitis were the result of infection, even if bacteria were not isolated from the pretreatment sample or from samples collected at the start of the study and those quarters were added to the totals, then reduction of new infection with the .1% product would be significant. Hoppe Seylers Z Physiol Chem, 1983 Dec, 364(12), 1777 - 93 Purification of Escherichia coli 30S ribosomal proteins by high performance liquid chromatography; Kamp RM et al.; High performance liquid chromatography was applied to the separation of proteins derived from the Escherichia coli 30S ribosomal subunit . Several methods of separating this protein mixture has been tested: size-exclusion chromatography on hydrophilic phases; ion exchange and reversed phase chromatography (on C2 to C18 hydrocarbon-bonded supports) . Various elution systems were examined in order to obtain pure proteins suitable for micro-sequence analysis . The resolution and yields of the proteins varied considerably, depending on the type of support and gradient system used . The best results were achieved with uniformly globular-shaped supports of large pore size, and by combining high performance size exclusion with rechromatography on reversed phase columns . Purification conditions for the individual proteins are listed . The methods employed avoid any precipitation step and allow easy identification of the proteins by one or two-dimensional gel electrophoresis, amino-acid analysis or direct manual or automatic micro-sequencing . Since the isolation time is much reduced compared with conventional purification procedures, the proteins obtained by the techniques described here are well suited for topographical and immunological studies or reconstitution assays . Ribosomal proteins of other organisms can be separated under similar conditions. Gann, 1983 Dec, 74(6), 829 - 36 Mechanism of inhibition of DNA polymerases by 4'-epiadriamycin and 4'-O-tetrahydropyranyladriamycin; Tanaka M et al.; 4'-Epiadriamycin and 4'-O-tetrahydropyranyladriamycin (THP-adriamycin), derivatives of adriamycin, strongly inhibited in vitro reactions of DNA polymerases alpha and beta from calf thymus by competing with activated DNA (template-primer) . Preincubation of DNA polymerases with 4'-epiadriamycin or THP-adriamycin strongly inhibited the DNA polymerase, but the inhibition was reversed by adding excess amounts of template-primer . These results indicate that 4'-epiadriamycin and THP-adriamycin inhibit the in vitro reactions of DNA polymerases by direct interaction of the drugs with the enzymes as well as by impairing the template activity through intercalation into DNA, in agreement with results obtained for other anthracycline antitumor agents, daunomycin and adriamycin . The activity of DNA polymerase alpha may be more sensitive to both 4'-epiadriamycin (Ki, 9 microM) and THP-adriamycin (Ki, 5.5 microM) than that of DNA polymerase beta (Ki 30 microM for 4'-epiadriamycin and 22 microM for THP-adriamycin) . DNA polymerase I from Escherichia coli was inhibited by these drugs in the same manner as DNA polymerase alpha . On the other hand, the inhibition of RNA polymerase from E . coli was more marked when the drug was preincubated with template DNA than with the enzyme. Eur J Clin Microbiol, 1983 Dec, 2(6), 564 - 7 Rapid bioluminescent assay for determining netilmicin and tobramycin concentrations in serum; Nilsson L; A rapid bioluminescent assay for determining netilmicin and tobramycin concentrations in serum based on the dose-dependent effect of these agents on the accumulation of extracellular ATP in Escherichia coli LU 14 cultures is presented . This strain of Escherichia coli is unaffected by antibiotics used in combination with aminoglycosides and it lacks significant ATP-ase activity, which is a prerequisite for extracellular ATP accumulation . ATP was quantified by the firefly bioluminescence system . The accuracy of the bioluminescent assay expressed as mean coefficient of variation over the therapeutic range was 3.2%; corresponding figures for EMIT and an agar disk diffusion assay were 4.2% and 4.8% respectively . All methods used correlated well (r = 0.935-0.986) when they were evaluated on clinical serum specimens . The bioluminescent assay requires 25 microliters serum and results are available within 75 min. Arch Microbiol, 1983 Dec, 136(4), 297 - 9 The complete sequence of murein synthesis in ether treated Escherichia coli; Metz R et al.; The in vitro synthesis of murein from the precursors UDP-N-acetylglucosamine, L-alanine, D-glutamic acid and meso-diaminopimelic acid was performed with the aid of ether treated Escherichia coli . This synthesis was sensitive to representative inhibitors of early reactions in the cytoplasm as well as of late reactions in the membrane or the cell wall . The sensitivity was higher than in in vitro systems starting with UDP-N-acetylmuramic acid or UDP-N-acetylmuramyl-pentapeptide. J Antimicrob Chemother, 1983 Dec, 12(6), 543 - 7 Nitrofuran reductase activity in nitrofurantoin-resistant strains of Escherichia coli K12: some with chromosomally determined resistance and others carrying R-plasmids; Breeze AS et al.; The nitrofuran reductase activity of Escherichia coli K12 strains was separated into two components by passage through Sepharose 4B-200 . The components differed in their sensitivity to 2 M urea and their reactivity with NADH and NADPH . Some nitrofurantoin-resistant mutants had lost one component (alpha) while retaining the other (beta) . Other mutants, resistant to higher levels of nitrofurantoin, had lost most of the beta component as well . Transconjugants which had received plasmids conferring nitrofurantoin resistance were found to have only the alpha component and it was markedly less active when NADH rather than NADPH was supplied as co-enzyme. J Antimicrob Chemother, 1983 Dec, 12(6), 535 - 42 Molecular basis of chloramphenicol and thiamphenicol toxicity to DNA in vitro; Skolimowski IM et al.; The action of thiamphenicol and reduced chloramphenicol on DNA has been investigated in vitro . Reduced chloramphenicol causes DNA damage which is dependent upon reduction of the nitro group and which is characterized by helix destabilization and strand breakage . Although the reduction process requires six electrons indicating formation of the amine in 100% yield the toxic agent is most probably a short-lived reduction intermediate . We propose the one-electron nitro radical anion rather than the nitroso derivative as the toxic agent responsible for DNA damage related to aplastic anaemia . In contrast, thiamphenicol produces no such effects on DNA. Genetika, 1983 Dec, 19(12), 2072 - 4 {Regulation of yeast gene Leu 2 expression in Escherichia coli by IS1 element}; Kashkin PK et al.; Expression of the yeast LEU2 gene in Escherichia coli is not sufficiently high . That is why the growth rate of leuB E . coli transformants slowed down on media lacking leucine . The mutations leading to the increase in yeast gene expression in bacteria were found . It has been shown that mutations are connected with insertion of the bacterial IS1 element into the regulatory region of LEU2 . The increase in the yeast gene expression does not depend on orientation of the inserted IS1 element . The IS1 element insertion seems to control the attenuation of transcription of LEU2. Genetika, 1983 Dec, 19(12), 1941 - 7 {Mutagenic and DNA-damaging effect of potassium bichromate in Escherichia coli cells}; Kalinina LM et al.; Mutagenic effects and DNA degradation in Escherichia coli cells treated with potassium bichromate were studied in this work . It is estimated that events taking place in cells treated with the mutagen are controlled by recB recC and sbcB gene products . The method for sedimentation analysis has revealed DNA degradation . The use of the impulse label of 3H-thymidine made it possible to detect DNA breaks in the daughter strands . This effect of potassium bichromate on DNA degradation is, possibly, connected with mutagenic repair. Biochem J, 1983 Dec 1, 215(3), 513 - 8 Chemical modification of Escherichia coli succinyl-CoA synthetase with the adenine nucleotide analogue 5'-p-fluorosulphonylbenzoyladenosine; Prasad AR et al.; Escherichia coli succinyl-CoA synthetase (EC 6.2.1.5) was irreversibly inactivated on incubation with the adenine nucleotide analogue 5'-p-fluorosulphonylbenzoyladenosine (5'-FSBA) . Optimal inactivation by 5'-FSBA took place in 40% (v/v) dimethylformamide . ATP and ADP protected the enzyme against inactivation by 5'-FSBA, whereas desulpho-CoA, an analogue of CoA, did not . Inactivation of succinyl-CoA synthetase by 5'-FSBA resulted in total loss of almost four thiol groups per alpha beta-dimer, of which two groups appeared to be essential for catalytic activity . 5'-FSBA at the first instance appeared to interact non-specifically with non-essential thiol groups, followed by a more specific reaction with essential thiol groups in the ATP(ADP)-binding region . Plots of the data according to the method of Tsou {(1962) Sci . Sin . 11, 1535-1558} revealed that, of the two slower-reacting thiol groups, only one was essential for catalytic activity . When succinyl-CoA synthetase that had been totally inactivated by 5'-FSBA was unfolded in acidic urea and then refolded in the presence of 100 mM-dithiothreitol, 85% of the activity, in comparison with the appropriate control, was restored . These data are interpreted to indicate that inactivation of succinyl-CoA synthetase by 5'-FSBA involves the formation of a disulphide bond between two cysteine residues . Disulphide bond formation likely proceeds via a thiosulphonate intermediate between 5'-p-sulphonylbenzoyladenosine and one of the reactive thiol groups of the enzyme. Antimicrob Agents Chemother, 1983 Dec, 24(6), 868 - 70 Inhibition of respiration of Escherichia coli by thioglycerol; Javor GT; Anaerobic growth on glucose significantly protected Escherichia coli from growth inhibition by thioglycerol . Methionine and anaerobiosis completely overcame growth inhibition by 2 to 90 mM thioglycerol . The respiration of aerobically growing cells was partially inhibited by 20 to 90 mM thioglycerol. Antimicrob Agents Chemother, 1983 Dec, 24(6), 860 - 7 Depression of adenosylmethionine content of Escherichia coli by thioglycerol; Javor GT; The mechanism of growth inhibition of Escherichia coli by thioglycerol was probed . The extent of growth inhibition depended on the ratio of thioglycerol to initial cell concentration . Exogenously added methionine and several methionine analogues reversed the inhibition of 2 to 40 mM thioglycerol . Exposure to thioglycerol elevated the intracellular concentration of methionine, but the level of S-adenosylmethionine was depressed . Thioglycerol was methylated in vivo to 3-methylthio-1,2-propanediol. Am J Vet Res, 1983 Dec, 44(12), 2391 - 4 Scanning electron microscopy of porcine jejunal loops infused with Escherichia coli endotoxin; Vogelweid CM et al.; Phenol-extracted Escherichia coli endotoxin was infused into ligated jejunal loops in 3 pigs . Tissue samples were collected from the center of each loop 6 hours later and examined with scanning electron microscopy . Control loops infused with pyrogen-free water were examined, as well as nonligated biopsy and blind-loop samples . Loops infused with endotoxin had predominantly ridge-shaped villi, many actively secreting goblet cells, and numerous large epithelial ulcerations involving the tips and sides of villi . Tight junction disruption between epithelial cells was observed in 1 pig . In contrast, small ulcers were rarely seen in ligated control loops . Villi were predominantly tongue-shaped, and goblet cell activity was moderate . Pathologic changes were greater in endotoxin-infused loops than in water-infused loops at 6 hours after infusion. Am J Vet Res, 1983 Dec, 44(12), 2373 - 5 Changes in penetrability of bovine papillary duct to endotoxin after milking; Schultze WD et al.; The relative penetrability of bovine papillary ducts was assayed using frequency of inflammatory response to implantation of Escherichia coli endotoxin at a depth of 3 mm . Penetrability immediately after machine milking, compared with penetrability after delays of 10 to 480 minutes, indicated a decrease in frequency of penetrability to a minimum at 2 hours after milking, a decrease of about 75% . Four and 8 hours after milking, frequency of penetrability increased to about 55% of that measured immediately after milking. Am J Vet Res, 1983 Dec, 44(12), 2262 - 7 Sequential response of milk leukocytes, albumin, immunoglobulins, monovalent ions, citrate, and lactose in cows given infusions of Escherichia coli endotoxin into the mammary gland; Guidry AJ et al.; Changes in concentrations of both the cellular and the humoral components of milk are known to occur during mastitis . This study was conducted to determine temporal changes in the concentrations of leukocytes, albumin, immunoglobulins (Ig), monovalent ions, lactose, and citrate in milk during the initial phases of simulated mastitis . Ten cows whose udders were pathogen free and had milk leukocyte counts of less than 0.5 X 10(6)/ml were used . Two dosages of Escherichia coli endotoxin were administered to simulate various degrees of mastitis . Two quarters in each cow were infused with the endotoxin and the other 2 served as controls . Quarter milk samples were collected frequently before and after infusion . Within 2 hours after infusion of a 100-micrograms dose of endotoxin, clinical mastitis was observed in most of the infused quarters . Leukocytes, albumin, IgG1, and conductivity showed significant increases . Values before infusion and at postinfusion (PI) hour 2 were as follows: leukocytes, 0.33 and 3.65 X 10(6)/ml, respectively; albumin, 0.38 and 4.49 mg/ml; IgG1, 0.34 and 0.79 mg/ml; and conductivity, 6.0 and 6.9 mmho . Average of the peak values and their average relative time of appearance after infusion were as follows: leukocytes, 28.82 X 10(6)/ml at 16 hours; albumin, 9.37 mg/ml at 4 hours; IgG1, 1.35 mg/ml at 4 hours; and conductivity, 95.5 mmho at 10 hours . The IgG1 values tended to remain high in the presence of rapidly declining albumin concentrations, indicating the possibility of an active, rather than a passive, transfer of IgG1 from the circulation . The response to the 10-micrograms dose of endotoxin ranged from subclinical to clinically mild mastitis with lesser cellular and humoral responses. Am J Vet Res, 1983 Dec, 44(12), 2221 - 5 Effect of salicylates on intestinal secretion in calves given (intestinal loops) Escherichia coli heat-stable enterotoxin; Wise CM et al.; The inhibitory effect of salicylates on intestinal secretion in 1- to 5-day-old calves given Escherichia coli heat-stable enterotoxin (ST)-induced intestinal fluid response was investigated . Purified ST was diluted in isotonic saline solution to obtain 1:10, 1:25, 1:50, 1:75, and 1:100 dilutions . Each dilution (1 ml) was inoculated into ligated loops in the distal part of the jejunum of each calf . Acetylsalicylic acid (aspirin) given orally (100 mg/kg) at 4 hours before ST was inoculated did not substantially alter the intestinal fluid response to ST . Sodium salicylate (IV) infusion, begun simultaneously when, or at 1 hour after, ST was inoculated, significantly (P less than 0.05) decreased fluid accumulation in those loops inoculated with ST dilutions of 1:25 or greater . The sodium and potassium concentrations of the accumulated fluid did not differ significantly between or within treatment groups . These results indicate that sodium salicylate infusion may be beneficial in treating enterotoxic colibacillosis in calves . Aspirin given orally at the dose used in the present study, would not have any beneficial effect. Anal Biochem, 1983 Dec, 135(2), 423 - 30 Detection of enzymatic activities in sodium dodecyl sulfate-polyacrylamide gels: DNA polymerases as model enzymes; Blank A et al.; Recent techniques for detecting the catalytic activity of enzymes in sodium dodecyl sulfate (SDS)-polyacrylamide gels have been hampered by lack of reproducibility associated with variability in commercial SDS preparations . Simple expedients which facilitate reproducible detection of DNA polymerase activity and which appear to be widely applicable to detection of other enzymes are reported here . It was observed that reproducibility of a reported procedure for DNA polymerase detection (Spanos, A., Sedgwick, S . G., Yarranton, G . T., Hubscher, U., and Banks, G . R . (1981) Nucl . Acids Res . 9, 1825-1839) depends on the SDS used for electrophoresis, and that sensitivity is markedly reduced if currently available SDS is substituted for the discontinued product specified by Spanos et al . A modified procedure yielding sensitivity with contemporary commercial SDS, which exceeds the sensitivity observed when using the protocol and the SDS originally specified, is described . The modifications employed, which presumably promote renaturation of enzymes, are (1) embedding fibrinogen in gels and (2) washing detergent from gels with aqueous isopropanol after electrophoresis . These expedients permit detection of picogram amounts of Escherichia coli DNA polymerase 1 and its Klenow fragment and nanogram amounts of calf thymus alpha and rat liver (Novikoff hepatoma) beta polymerases . Finally, it is shown that sensitivity of DNA polymerase detection is reduced by lipophilic contaminants in contemporary commercial SDS, and that the expedients employed here mitigate the deleterious effect of these impurities. Radiat Res, 1983 Dec, 96(3), 635 - 40 Mutation by {5-3H}cytosine decay in DNA of Escherichia coli lacking uracil-DNA glycosylase activity; Bockrath R et al.; The mutagenic local effect of tritium decay at the 5 position of cytosine in DNA of Escherichia coli was determined in wild-type and in ung strains defective in uracil-DNA glycosylase . In the absence of this in vivo activity any genetic consequences of uracil residues formed in DNA should be enhanced . However, the mutation frequency response was no greater in the mutant strain than in the wild type . This finding is inconsistent with the earlier suggestion that efficient production of C to T transitions by the local effect of {5-3H}cytosine decay results from the formation of uracil in cellular DNA . Some other intermediate should be considered, one that is not a substrate for uracil-DNA glycosylase. Lab Invest, 1983 Dec, 49(6), 716 - 24 Role of neutrophils in the deposition of platelets during acute inflammation; Issekutz AC et al.; Platelets can release a variety of inflammatory mediators . These formed elements have been shown to accumulate early in acute inflammation, often prior to fibrin and thrombus formation . Polymorphonuclear leukocyte (PMNL) infiltration is also an early event and is linked to protein exudation and hyperemia . The relationship between PMNL and platelet accumulation was studied . Inflammation was induced in rabbits by intradermal injection of stimuli, and 51Cr-labeled leukocytes and 111In-labeled platelets were used to quantitate the rates of leukocyte and platelet accumulation in the dermal reactions . A temporal association between the rates of leukocyte infiltration (greater than 95% PMNL) and of platelet deposition at skin sites was observed when killed Escherichia coli, E . coli-derived chemotactic factors, zymosan-activated plasma, f-met-leu-phe, or endotoxin were injected intradermally . A linear correlation (r = 0.96; p less than 0.001) between these parameters was observed . 59Fe-labeled red cells did not accumulate in these lesions . Platelet deposition in response to inflammatory stimuli did not occur in PMNL-depleted (nitrogen mustard treatment) but platelet-sufficient rabbits, in spite of normal platelet deposition in thrombin-injected sites . Platelet responses to inflammatory stimuli were normal when neutropenia, after nitrogen mustard treatment, was prevented . In contrast, in situ PMNL reconstitution of the skin sites in neutropenic rabbits did not cause local platelet accumulation . Intravenous infusion of the synthetic chemotactic factor, f-met-leu-phe, induced transient neutropenia and thrombocytopenia (30% of control) with platelet sequestration primarily in the lung . This is also the known site of PMNL sequestration during f-met-leu-phe infusion . It is concluded that platelets selectively deposit in acutely inflamed tissues primarily during PMNL margination in, and emigration across, the microvasculature . Platelets and PMNLs likely coassociate on the vessel wall, and this interaction may influence the course of inflammation. J Trauma, 1983 Dec, 23(12), 1030 - 3 Expanded microporous polytetrafluoroethylene (PTFE) grafts in contaminated wounds: experimental and clinical study; Shah PM et al.; Autogenous vein graft is regarded as an ideal arterial substitute for its long-term patency and relative resistance to infection . A clinical instance of life-threatening hemorrhage from an infected disrupted vein graft stimulated a study in dogs, comparing vein and PTFE graft performance in wounds contaminated with S . aureus and E . coli cultured from the patient's wound . Infective disruption of vein wall occurred in three of ten animals resulting in exsanguination and death . Host artery disruption at PTFE suture line occurred in one of ten animals . Thrombosis of graft and host artery in this animal precluded hemorrhage and death . This led to favorable clinical experiences with PTFE grafts in contaminated wounds of 22 trauma patients . It is concluded that PTFE is better assurance against disruption and hemorrhage than vein graft in contaminated, potentially infected sites . PTFE may be used preferentially as a vascular substitute in trauma patients provided that all traditional surgical safeguards and principles are followed. J Infect Dis, 1983 Dec, 148(6), 1114 - 9 Protection of suckling mice from the heat-stable enterotoxin of Escherichia coli by human milk; Cleary TG et al.; Human milk contained a factor or factors that protected suckling mice from lethal fluid loss due to Escherichia coli heat-stable toxin . This protective effect was present throughout lactation . The factor was heat stable, acid and alkali stable (pH 2 and 12), and of low molecular weight . Ion-exchange chromatography indicated that it was uncharged at neutral pH . The factor was insensitive to proteinase K digestion and partitioned in the aqueous phase in 2:1 (vol/vol) chloroform-methanol . Acid hydrolysis (2 N HCl at 100 C for 3 hr) destroyed the protective effect . Neither an amino acid mix simulating human milk nor lactose in phosphate-buffered saline was protective . These data indicate that the protective factor in human milk is neither a lipid nor a protein but appears to be a carbohydrate. J Clin Microbiol, 1983 Dec, 18(6), 1429 - 31 Identification of enterotoxigenic Escherichia coli isolated from swine with diarrhea in Thailand by colony hybridization, using three enterotoxin gene probes; Patamaroj U et al.; The DNA colony hybridization assay was used to identify enterotoxigenic Escherichia coli among E . coli isolated from 803 swine with diarrhea at 10 farms in Thailand . Between 5 September and 8 December 1981, enterotoxigenic E . coli were identified in 40% of 58 litters of piglets under 10 days old and 17% of 29 litters between 10 and 21 days old with diarrhea at farms at four different locations in Thailand . All E . coli that hybridized with one or more of the three enterotoxin gene probes produced heat-labile or heat-stable toxin or both, as determined by testing culture supernatants in the Y1 adrenal and suckling mouse assays . The DNA colony hybridization technique is a specific method of identifying enterotoxigenic E . coli from swine and can be used to further characterize these enteric pathogens. J Clin Microbiol, 1983 Dec, 18(6), 1413 - 6 Increased frequency of ColV plasmids and mannose-resistant hemagglutinating activity in an Escherichia coli K1 population; Aguero ME et al.; The expression of traits linked to pathogenicity was studied in a population of Escherichia coli K1 strains . It was found that E . coli K1 strains isolated from extraintestinal infection harbor the ColV plasmid and express mannose-resistant hemagglutinating activity type VI with a high frequency . The presence of these properties may play a role in the ability of some E . coli K1 serogroups to invade. Eur J Biochem, 1983 Dec 1, 137(1-2), 337 - 45 Distance measurement by energy transfer . Ribosomal proteins L6, L10 and L11 of Escherichia coli; Steinhauser KG et al.; Ribosomal proteins L6, L11 and the complex {(L12)4 X L10} were labelled specifically at their respective single thiol groups, either with the acetylaminoethyl-dansyl or with the acetamidofluorescein fluorophore . The labelled proteins were then reconstituted, singly or in pairs, into ribosomal 50S subunits; the presence of the label had no observable effect on the composition, shape or activity of the reconstituted subunits . The distances between the labelled thiol groups were measured by a fluorescence energy transfer method detailed elsewhere {Epe, B . et al . (1983) Proc . Natl Acad . Sci . USA, 80, 2579-2583} and were found to be: for L6-L10, 60 A (6.0 nm); for L6-L11, 46 A (4.6 nm); for L10-L11, 56 A (5.6 nm) . Reversal of the direction of energy transfer by exchanging labels gave duplicate distances which differed, on average, by about 4% . The distance between the fluorescent labels on L10 and L11 in the {23 S-RNA X L10 X L11 X (L12)4} ribonucleoprotein complex was the same as in the 50S subunit, but all three distances were greater in 50S subunits which had been reconstituted without the final activation step (incubation at 50 degrees C) . This suggests a tightening of the L6/L10/L11 domain of the 50S subunit during the activation step. Eur J Biochem, 1983 Dec 1, 137(1-2), 279 - 92 Sequence-specific resonance assignments in the 1H nuclear-magnetic-resonance spectrum of the lac repressor DNA-binding domain 1-51 from Escherichia coli by two-dimensional spectroscopy; Zuiderweg ER et al.; The assignment of the 1H nuclear magnetic resonance (NMR) spectrum of the DNA-binding domain 1-51 of lac repressor from Escherichia coli is described and documented . The assignments are based entirely on the amino acid sequence and on two-dimensional NMR experiments at 360 MHz and 500 MHz . Individual assignments were obtained at 18 degrees C for the backbone protons of 44 out of the total of 51 amino acids residues, the exceptions being Met-1, Lys-2, Tyr-7, Arg-35, Glu-36, Lys-37 and Ile-48 . Complete assignments of the non-labile hydrogen atoms of the side chain were obtained for 33 residues, and for Asn-46 and Asn-50 the delta amide protons were also identified . The chemical shifts for the assigned resonances at 18 degrees C are listed for an aqueous solution at pH 4.9 and at pH 6.8. Carcinogenesis, 1983 Dec, 4(12), 1591 - 7 Mechanisms of chemical mutagenesis and carcinogenesis: effects on DNA replication of methylation at the O6-guanine position of dGTP; Toorchen D et al.; The incorporation of O6-methyl-dGTP during DNA replication in vitro by 'Klenow' E . coli pol I was determined . O6-Methyl-dGTP was found to: (i) incorporate opposite T and C template residues, with a greater than 20-fold preference for T, and (ii) arrest DNA synthesis when incorporated in place of dATP at all but pyrimidine-rich growing-strand sequences . The significance of O6-methyl-dGTP incorporation during DNA biosynthesis in vivo for mutagenesis and carcinogenesis is discussed. Proc Natl Acad Sci U S A, 1983 Dec, 80(23), 7085 - 9 Identification of the epsilon-subunit of Escherichia coli DNA polymerase III holoenzyme as the dnaQ gene product: a fidelity subunit for DNA replication; Scheuermann R et al.; Based on extensive genetic and biochemical studies, the multisubunit DNA polymerase III holoenzyme is considered responsible for the chain-elongation stage in replication of the genome of Escherichia coli and is thus expected to be the major determinant of fidelity as well . Previous experiments have shown that two mutations conferring a very high mutation rate on E . coli, mutD5 and dnaQ49, decrease severely the 3' leads to 5' exonucleolytic editing activity of the polymerase III holoenzyme . To identify more precisely the nature of these mutations, we have carried out genetic mapping and complementation experiments . From these studies and experiments by others, we conclude that the most potent general mutator mutations in E . coli occur in a single gene, dnaQ . To define further the role of the dnaQ gene, we have used two-dimensional gel electrophoresis to compare the labeled dnaQ gene product with purified polymerase III holoenzyme . The dnaQ product comigrates with the epsilon-subunit, a 25-kilodalton protein of the polymerase III "core" enzyme . We conclude that the epsilon-subunit of polymerase III holoenzyme has a special role in defining the accuracy of DNA replication, probably through control of the 3' leads to 5' exonuclease activity. Nature, 1983 Dec 1-7, 306(5942), 441 - 7 Modular arrangement of functional domains along the sequence of an aminoacyl tRNA synthetase; Jasin M et al.; Gene deletions show that much of Escherichia coli alanine tRNA synthetase is dispensable for each of three activities and that these activities appear to require specific domains arranged linearly along the polypeptide . Thus, variable fusions of extra polypeptide domains to a catalytic core may account for the diverse of aminoacyl tRNA synthetases. J Bacteriol, 1983 Dec, 156(3), 1359 - 62 In vitro coupled transcription-translation of linear DNA fragments in a lysate derived from a recB rna pnp strain of Escherichia coli; Bassett CL et al.; A recB21 derivative (CLB7) of an Escherichia coli rna-19 pnp-7 strain (PR7) was constructed for use in examining the in vitro coupled transcription-translation of linear DNA . The expression of linearized DNAs in CLB7 (recB21 rna-19 pnp-7) lysates was enhanced significantly when compared with expression of the same DNAs in lysates prepared from the PR7 or the original recB21 (CF300) strains . In addition, the endogenous incorporation of {35S}methionine into protein was considerably reduced in CLB7 lysates relative to lysates derived from the original recB21 strain. J Bacteriol, 1983 Dec, 156(3), 1275 - 81 Effect of pKM101 on cell killing and specificity of mutation induction by cis-diaminedichloroplatinum(II) in Escherichia coli K-12; Brouwer J et al.; Cell killing and mutation induction in the lacI gene of Escherichia coli by cis-Pt(NH3)2Cl2 were studied in cells with different repair capacities, with and without pKM101 . The presence of the plasmid pKM101 made repair-proficient cells more susceptible to killing by cis-Pt(NH3)2Cl2 and strongly enhanced mutation induction by that compound . Both effects were shown to be dependent upon excision repair . Characterization of the induced mutations in the lacI gene after cis-Pt(NH3)2Cl2 treatment of E . coli cells, by the LacI system, revealed that the mutagenic specificity of the Pt compound was strongly influenced by the presence of the pKM101 plasmid . With pKM101, 23% of the induced amber and ochre mutations resulted from substitutions at AT base pairs, whereas these mutations were hardly induced in cells without pKM101 . These results suggest that pKM101-induced repair differs from normal SOS repair. J Bacteriol, 1983 Dec, 156(3), 1268 - 74 Limited homology between trg and the other transducer proteins of Escherichia coli; Engstrom P et al.; Transducers are transmembrane proteins that are central to the chemotactic system of Escherichia coli . The proteins transduce ligand recognition into an excitatory signal and function in adaptation as methyl-accepting proteins . The transducer genes tsr, tar, and tap have extensive homology with each other . However, previous studies revealed little indication of homology between those three transducer genes and a fourth gene, trg . We investigated the relationship between trg and the other genes by blot-hybridization experiments and the relationship between Trg and the other transducer proteins by immune precipitation and experiments with an antiserum raised to purified Trg protein . In experiments in which 35% mismatch would be tolerated, weak hybridization of trg was detected to a DNA fragment containing tar and tap but not to a fragment containing tsr . In experiments in which only 30% mismatch would be tolerated, no trg hybridization was apparent either to total chromosomal DNA or to DNA from hybrid plasmids carrying the other transducer genes . An anti-Trg serum formed immune precipitates with the Tsr and Tar proteins as well as with the Trg protein to which it was raised . We conclude that there is homology between Trg and the other transducer, but the homology is more limited than that shared among the other transducers . Furthermore, we found no indication of additional transducer genes closely related to trg . Thus, the trg gene is a somewhat distant cousin within a single transducer gene family of E . coli. J Bacteriol, 1983 Dec, 156(3), 1228 - 35 Requirement of the cheB function for sensory adaptation in Escherichia coli; Yonekawa H et al.; The chemotactic behavior of Escherichia coli mutants defective in cheB function, which is required to remove methyl esters from methyl-accepting chemotaxis proteins, was investigated by subjecting swimming or antibody-tethered cells to various attractant chemicals . Two cheB point mutants, one missense and one nonsense, exhibited stimulus response times much longer than did the wild type, but they eventually returned to the prestimulus swimming pattern, indicating that they were not completely defective in sensory adaptation . In contrast, strains deleted for the cheB function showed no evidence of adaptation ability after stimulation . The crucial difference between these strains appeared to be the residual level of cheB-dependent methylesterase activity they contained . Both point mutants showed detectable levels of methanol evolution due to turnover of methyl groups on methyl-accepting chemotaxis protein molecules, whereas the cheB deletion mutant did not . In addition, it was possible to incorporate the methyl label into the methyl-accepting chemotaxis proteins of the point mutants but not into those of the cheB deletion strain . These findings indicate that cheB function is essential for sensory adaptation in Escherichia coli. J Bacteriol, 1983 Dec, 156(3), 1093 - 8 Mechanisms for recF-dependent and recB-dependent pathways of postreplication repair in UV-irradiated Escherichia coli uvrB; Wang TC et al.; The molecular mechanisms for the recF-dependent and recB-dependent pathways of postreplication repair were studied by sedimentation analysis of DNA from UV-irradiated Escherichia coli cells . When the ability to repair DNA daughter strand gaps was compared, uvrB recF cells showed a gross deficiency, whereas uvrB recB cells showed only a small deficiency . Nevertheless, the uvrB recF cells were able to perform some limited repair of daughter strand gaps compared with a "repairless" uvrB recA strain . The introduction of a recB mutation into the uvrB recF strain greatly increased its UV radiation sensitivity, yet decreased only slightly its ability to repair daughter strand gaps . Kinetic studies of DNA repair with alkaline and neutral sucrose gradients indicated that the accumulation of unrepaired daughter strand gaps led to the formation of low-molecular-weight DNA duplexes (i.e., DNA double-strand breaks were formed) . The uvrB recF cells were able to regenerate high-molecular-weight DNA from these low-molecular-weight DNA duplexes, whereas the uvrB recF recB and uvrB recA cells were not . A model for the recB-dependent pathway of postreplication repair is presented. J Bacteriol, 1983 Dec, 156(3), 1019 - 24 Citrate utilization by Escherichia coli: plasmid- and chromosome-encoded systems; Reynolds CH et al.; Citrate utilization plasmids have previously been identified in atypical Escherichia coli isolates . A different citrate-utilizing (Cit+) variant of E . coli K-12 arose as a consequence of two chromosomal mutations (B . G . Hall, J . Bacteriol . 151:269-273, 1982) . The processes controlling the transport of citrate in both a Cit+ chromosomal mutant and a Cit+ plasmid system were studied . Both systems were found to be inducible in growth experiments . In transport assays with whole cells, citrate-grown cells accumulated {1,5-14C}citrate at two to three times the rate of uninduced cells . Only the Vmax was affected by induction, and the Km for whole cells remained at 67 microM citrate for the chromosomal strain and 120 microM citrate for the plasmid-conferred system . There was no detectable accumulation of radioactivity with {6-14C}citrate, because of rapid metabolism and the release of 14CO2 . Energy-dependent citrate transport was found with membrane vesicles obtained from both the chromosome-conferred and the plasmid Cit+ systems . The vesicle systems were inhibited by valinomycin and carbonyl cyanide m-chloro-phenylhydrazone but not by nigericin and monensin . In contrast to whole cells, the vesicle systems were resistant to Hg2+ and showed identical kinetics with {1,5-14C}citrate and {6-14C}citrate . H+ appeared to be important for citrate transport in whole cells and membranes . Monovalent cations such as Na+ and K+, divalent cations such as Mg2+ and Mn2+, and anions such as PO4(3-), SO4(2-), and NO3- were not required . The two systems differed in inhibition by citrate analogs. Genetics, 1983 Dec, 105(4), 829 - 42 Characterization of the operator sites of the exu regulon in Escherichia coli K-12 by operator-constitutive mutations and repressor titration; Mata-Gilsinger M et al.; In Escherichia coli, the exu regulon of the hexuronate system involves the three exuT, uxaCA and uxaB operons and is under the negative control of the exuR regulatory gene product . The technique developed by Casadaban, Chou and Cohen was employed to construct two plasmids containing operon fusions in which the lactose genes were fused to the uxaCA and exuT operons . These fusions were transferred into the chromosome by a reciprocal recombination event, and the resulting strains were used for isolation of mutants defective in repression . Two types of operator-constitutive mutants were obtained: one specific for the uxaCA operon expression and the other affecting the exuT gene expression . This genetic evidence confirms that these two operons which are divergently transcribed each possess their own operator site.--The derepressed expression of the two exuT-lac and uxaCA-lac operons and the uxaB gene was also examined upon introduction of plasmids bearing various operators of the exu regulon . The results of testing exuR repressor titration by multiple copies of the exu operators allowed us to show a gradation in the affinity degrees for the three exu operators: uxaBo has the strongest affinity for the exuR repressor and uxaCo the weakest, although that of exuTo seems to be just slightly greater . This gradation may play a role in the control of the exu regulon expression. Gene, 1983 Dec, 26(2-3), 295 - 9 Expression in Streptomyces ambofaciens of an Escherichia coli K-12 gene which confers resistance to hygromycin B; Kuhstoss S et al.; We have constructed two plasmid vectors (pKC293 and pKC305) that can replicate in Escherichia coli K-12 and Streptomyces ambofaciens . These shuttle vectors were used to demonstrate the expression of two E . coli genes, hygromycin B (Hm) resistance and Tn5 neomycin (Nm) resistance, in S . ambofaciens. Gene, 1983 Dec, 26(2-3), 197 - 203 Tetracycline resistance determined by pBR322 is mediated by one polypeptide; Backman K et al.; Only one polypeptide specified by plasmid pBR322 is necessary to determine tetracycline resistance . Small deletions in pBR322 constructed in vitro which result in the lack of ability to confer tetracycline resistance in vivo also result in the absence or alteration of this polypeptide in vivo . Other deletions define the extent of material necessary to encode this polypeptide . A correction to the DNA sequence of the tetracycline resistance cistron has been determined which confirms these observations. Gene, 1983 Dec, 26(2-3), 171 - 9 Lambda Charon vectors (Ch32, 33, 34 and 35) adapted for DNA cloning in recombination-deficient hosts; Loenen WA et al.; New phage lambda-based cloning vectors, Charons 32, 33, 34 and 35, have been constructed . These vectors allow cloning of large (19-21 kb) DNA fragments in up to six cloning sites . DNA cloned in these vectors can be propagated on recA- Escherichia coli hosts. Gene, 1983 Dec, 26(2-3), 137 - 46 Double cos site vectors: simplified cosmid cloning; Bates PF et al.; A new vector for construction of cosmid libraries is described . Cosmid c2XB contains restriction sites for use in the insertion of foreign DNA and two lambda cos sites separated by a blunt-end restriction site . The presence of two cos sites on a single plasmid eliminates the need to prepare two separate cosmid arms, and the internal blunt-end restriction site prevents cosmid concatemerization . Thus, a double restriction-enzyme digestion is sufficient to prepare the vector for subsequent ligation with DNA fragments which are dephosphorylated to prevent their self-ligation . The use of this vector system allows efficient cosmid cloning (1 X 10(5) colonies per micrograms insert DNA) and eliminates background due to vector self-ligation . Furthermore, the procedure is so rapid as to eliminate the need to amplify cosmid libraries for storage and reuse . Also described is a cosmid vector for use in construction of cosmid libraries which are to be introduced into cultured eukaryotic cells . This vector contains the Herpes simplex virus thymidine kinase (HSV tk) gene as a selectable marker and a retroviral long terminal repeat (LTR) region as an enhancer sequence. Gene, 1983 Dec, 26(1), 25 - 39 Clone banks of cDNA from the parasite Schistosoma mansoni: isolation of clones containing a potentially immunodiagnostic antigen gene; Cordingley JS et al.; We have constructed a small vector specifically for blunt-end cloning of fragments of DNA . Both the PvuII site and the EcoRI site allow the detection of recombinants using a simple and inexpensive colour screen . We have used this vector to construct cDNA clone banks from polyadenylated messenger RNA {poly(A)+mRNA} from several life cycle stages of the human parasite Schistosoma mansoni and have identified clones encoding an immunodiagnostic antigen gene by a combination of Southern blotting and mRNA hybrid-selection and in vitro translation . Antibodies against this antigen are only present in patients infected with S . mansoni. Gene, 1983 Dec, 26(1), 19 - 24 Studies on the SV40-like papovavirus SV40-GBM . II . Molecular cloning in Escherichia coli of variant DNA molecules from glioblastoma-derived virus; Krause H et al.; The complete DNA genomes of the SV40-like GBM virus (GBM1), isolated from a human glioblastoma multiforme, and of two discrete classes of GBM DNA molecules that appear following three passages in CV-1 monkey cells at low multiplicities (GBM3-H and GBM3-L), were cloned in Escherichia coli using plasmid vector pBR322 . The cloned viral DNAs were characterized (i) by digestion of the chimeric plasmid DNAs with various restriction enzymes followed by comparison of their electrophoretic mobilities in agarose with that of similarly digested uncloned DNA, (ii) by hybridization of digested chimeric plasmid DNAs to 32P-labeled uncloned GBM DNA, and (iii) by electron microscopy . In restriction enzyme analysis the cloned GBM DNAs showed the same cleavage pattern as the uncloned DNAs, indicating that no major insertions or deletions were present . The electrophoretic data were confirmed by electron microscopic heteroduplex analysis. Gene, 1983 Dec, 26(1), 11 - 8 Overproduction of Escherichia coli NusA protein; Olins PO et al.; The nusA gene of Escherichia coli has been cloned into the plasmid vector pKC30 under the control of the inducible lambda pL promoter . When a strain carrying this plasmid is induced, NusA protein is overproduced more than 100-fold and constitutes 20-30% of the total cellular protein . The NusA protein purified from this strain appears identical to authentic NusA protein in its migration on SDS polyacrylamide gels and on isoelectric focusing gels . It is also able to function properly in in vitro termination and antitermination assays and in its ability to bind to E . coli core RNA polymerase. Can J Biochem Cell Biol, 1983 Dec, 61(12), 1315 - 21 Identification and physical characterization of a Col E1 hybrid plasmid containing a catalase gene of Escherichia coli; Loewen PC et al.; A hybrid Escherichia coli: Col E1 plasmid, pLC36-19, containing a catalase gene has been identified in the Clarke and Carbon colony bank . Catalase activity was amplified two- to three-fold in the pLC36-19-containing strain relative to other hybrid-plasmid-containing strains and this activity could be induced three- or four-fold by hydrogen peroxide or ascorbic acid . The plasmid was transferred to a strain chromosomally deficient in catalase synthesis, resulting in a strain with high and inducible levels of catalase . The plasmid was also transferred to a minicell-producing strain and minicells harbouring the plasmid were found to synthesize a labelled protein with a molecular weight of 84 000 characteristic of catalase from E . coli . A catalase activity was also synthesized by the plasmid-containing minicells . Two catalase activities with associated peroxidase activities coded for by the plasmid were separable by polyacrylamide gel electrophoresis and migrated coincident with chromosomally encoded catalase-peroxidase activities . A third catalase activity which did not have an associated peroxidase activity was not coded for by the plasmid . A physical map of the 25.5-kilobase pair plasmid was constructed by restriction nuclease analysis and the relative positions of 38 restriction sites were defined. Arch Biochem Biophys, 1983 Dec, 227(2), 596 - 608 Intracistronic mapping of the defective site and the biochemical properties of beta subunit mutants of Escherichia coli H+-ATPase: correlation of structural domains with functions of the beta subunit; Kanazawa H et al.; Sixteen mutants of Escherichia coli defective in H+-ATPase (proton-translocating ATPase) were tested for their ability to recombine with hybrid plasmids carrying various portions of the beta subunit cistron . Twelve mutations were mapped within the carboxyl half of the cistron corresponding to amino acid residues 279 to 459 (domain II), while four mutations were mapped within residues 17 to 278 (domain I) . The biochemical properties of these mutants were analyzed in terms of the proton permeability of their membranes and the assembly properties of their F1F0 complex . The mutants were classified according to the properties into three types, I, II, and III . In 12 mutants of type I, proton conduction in membrane vesicles was blocked and little F1 was released from the membranes under conditions in which F1 could be released from wild-type membranes, suggesting that assembly of the F1F0 complex is structurally and functionally defective . F1 was partially purified with very low recovery from one of the type I mutants, KF16 . ATPase activity was reconstituted from this F1 with the beta subunit of the wild type, confirming the genetic results . Only one mutant, KF38, was classified as type II . Its membranes were partially leaky to protons and its F1 was releasable, suggesting that the interaction of its F1 and F0 was unstable . Type III mutants, KF11 and KF43, had an F1F0 complex with very low activity, in which the structure of F1 was relatively similar to that of the wild type . F1 was purified as a single complex from KF43 in this study and from KF11 previously (H . Kanazawa, Y . Horiuchi, M . Takagi, Y . Ishino, and M . Futai (1980) J . Biochem . 88, 695-703) . Reconstitution experiments in vitro showed that the F1's of both mutants were defective in the beta subunit . The properties of the altered F1 of KF43 differed from those of F1 of KF11, suggesting that the mutation sites of KF43 and KF11 were different . From the results of mapping mutation sites and the biochemical properties of the mutants, the correlation of structural domains with function of the beta subunit is discussed . Most type I and type II mutations except that of KF39 were mapped in domain II, while the type III mutations were mapped in domain I, suggesting that domain II is more important than domain I for the function of the beta subunit, especially in terms of proper assembly of the F1F0 complex. J Antibiot (Tokyo), 1983 Dec, 36(12), 1743 - 7 Extraction, cloning and physical maps of plasmid DNAs from Streptomyces noursei; Takeda K et al.; Three plasmids, pSCY 2, pSCY 3 and pSCY 4, were detected in Streptomyces noursei B3, a producer of cycloheximide and nystatin . pSCY 3 and pSCY 4 had molecular sizes of 15.1 megadaltons (Md) and 3.2 Md, respectively . When covalently closed circular (ccc) DNAs were extracted from the mycelia during the course of cultivation, the yield of ccc DNA extracted per mycelium reached the highest value at the starting point of the exponential growth phase and decreased rapidly during exponential phase; the yield increased gradually in stationary phase . The smallest plasmid, pSCY 4, could not be detected after 42 hours of cultivation . To purify and characterize DNA of the major plasmid pSCY 3, it was cloned into Escherichia coli using pACYC 184 as a vector, and the physical maps of the composite plasmids were constructed. Anal Biochem, 1983 Dec, 135(2), 416 - 22 Isolation of DNA from filamentous fungi and separation into nuclear, mitochondrial, ribosomal, and plasmid components; Garber RC et al.; A general procedure for purifying and efficiently separating four types of DNA from filamentous fungi has been developed . The protocol involves (i) disruption of mycelial cells by blending in liquid nitrogen followed by suspension of cell contents in buffer containing high concentrations of protease and EDTA; (ii) deproteinization with phenol; (iii) cesium chloride/bisbenzimide density gradient centrifugation to separate nuclear DNA, mitochondrial DNA, and ribosomal DNA; and (iv) agarose gel electrophoresis to identify and purify plasmid DNA, if present . All DNAs are suitable for digestion with restriction endonucleases, ligation, and cloning in Escherichia coli, and DNAs from step three are recovered in high-molecular-weight form . The procedure has been used successfully with several dozen isolates of the plant pathogenic fungus Cochliobolus heterostrophus (including both laboratory strains and isolates collected directly from the field), and has been found to be equally suitable for C . carbonum, Neurospora crassa, N . tetrasperma, and Nectria haematococca. Anal Biochem, 1983 Dec, 135(2), 345 - 8 A simple and rapid procedure for the purification of plasmid DNA using reverse-phase C18 silica beads; Sparks RB et al.; A simple and efficient procedure for the rapid isolation of plasmid DNA free of chromosomal DNA and with only minor contamination with RNA is described . The protocol is a modification of the boiling method described by Holmes and Quigley {(1981) Anal . Biochem . 114, 193-197.} and utilizes C18 reverse-phase silica beads for final concentration and purification of plasmid DNA . The entire procedure can be carried out in 1 day and does not require the use of phenol or cesium chloride gradients, which require considerable labor and may sometimes cause nicking and lower recoveries of supercoiled DNA . The plasmid DNA obtained by this method retains biological activity, is supercoiled, and is suitable for restriction and DNA sequence analysis. Anal Biochem, 1983 Dec, 135(2), 280 - 7 Superoxide dismutase assay using alkaline dimethylsulfoxide as superoxide anion-generating system; Hyland K et al.; Alkaline dimethylsulfoxide as a superoxide anion-generating system in association with cytochrome c as a superoxide anion-indicating scavenger has been used to develop a new assay for superoxide dismutase . The assay is sensitive (one unit of enzymatic activity is provided by 110 ng of purified copper-containing superoxide dismutase) and highly specific . The nature of this system prevents the usual interferences and its simplicity allows for multiple, rapid measurements of superoxide dismutase activity in biological preparations using either normal or automated procedures. Eur J Biochem, 1983 Dec 1, 137(1-2), 263 - 7 Defective complex formation between single-stranded DNA and mutant recA430 or recA1 protein of Escherichia coli; Wabiko H et al.; The formation of a complex between recA protein and single-stranded DNA (ssDNA) was studied by filter-binding assays and sucrose density gradient centrifugation . In the presence of excess ATP, the wild-type recA protein formed salt-resistant complexes with ssDNA . One mutant recA430 (lexB30) protein bound to ssDNA slightly less effectively than wild-type protein, and the complexes had lost the stability to salt . Another mutant recA1 protein did not form complexes with ssDNA . On the other hand, in the absence of ATP, all proteins bound to ssDNA with the same efficiency, but all of the complexes were unstable in the presence of salt . The hybridization reaction in which homologous ssDNA is converted to double-stranded DNA (dsDNA) catalyzed by the recA430 protein was more sensitive to salt than that catalyzed by the wild-type protein . No hybridization reaction was found with the recA1 protein. Cell, 1983 Dec, 35(2 Pt 1), 511 - 20 Insertions, deletions and mismatches in heteroduplex DNA made by recA protein; Bianchi ME et al.; E . coli recA protein promotes the pairing of circular single strands with linear duplex DNA and the subsequent formation of large heteroduplex joints . From fd and M13 DNA, recA protein can make heteroduplex joints that include every kind of single base-pair mismatch . Aided by E . coli single-strand binding protein (SSB) and ATP regeneration, recA protein can incorporate into heteroduplex DNA insertions that are hundreds of base pairs long, whether the extra DNA is located initially in either the single-stranded or the double-stranded substrate . The ability of recA protein to span large insertions in the duplex DNA indicates that it unwinds a sizeable number of turns in advance of the growing heteroduplex joint . These observations show that an enzymatic basis exists in E . coli for forming extensively mismatched heteroduplex DNA, which might be involved in conversion-like events associated with recombination. Cell, 1983 Dec, 35(2 Pt 1), 495 - 502 DNA-protein interaction at the origin of DNA replication of the plasmid pSC101; Vocke C et al.; The initiation of DNA replication of the low copy number plasmid pSC101 is dependent on the dnaA initiator protein encoded by Escherichia coli . We have previously reported that the minimum essential replicon of the plasmid encodes a approximately 37.5 kd protein and that the protein is necessary along with host-encoded proteins for the replication of the plasmid chromosome . In this communication we show that the plasmid-encoded protein has sequence-specific DNA-binding activity . The protein binds cooperatively to the replication origin of pSC101 . Using chemical and enzymatic probes we have determined the contact points of the protein with the DNA and the precise domain of the replication origin recognized by the 37.5 kd protein . The specificity of the DNA-protein interaction would suggest that the 37.5 kd protein may possibly function by guiding the replisome to the correct DNA sequence on the chromosome of pSC101. Biol Reprod, 1983 Dec, 29(5), 1319 - 26 Testicular macrophages: isolation, characterization and hormonal responsiveness; Yee JB et al.; Macrophages were isolated from rat testes with trypsin treatment and established in culture using a differential attachment technique . The cells were maintained in culture in Medium 199 at 32 degrees C . The cells were then characterized for their ability to express traditional immunological function as well as to secrete lactate under the